Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhea, the more serious infections pelvic inflammatory disease (PID) and disseminated gonococcal infection (DGI). We identified a 57 kb genetic island present in N. gonorrhoeae that shows the characteristics of a pathogenicity island. Several forms of the gonococcal genetic island (GGI) are present among disease isolates, with approximately 80 percent of gonococcal strains carrying some form of the GGI. We identified a peptidoglycan hydrolase (atlA) encoded in the GGI and found that it is involved in the production of an unusual cytotoxin derived from the bacterial cell wall. This peptidoglycan-derived cytotoxin (PG-cytotoxin) is identical to the tracheal cytotoxin of Bordetella pertussis and is an important virulence factor in gonococcal infections. PG-cytotoxin causes the death of ciliated fallopian tube cells in organ culture, induces arthritis in rats, and induces IL-1 and IL-6 in cultured cells. The presence of atlA in the genetic island is significantly correlated with strains that cause DOT. DNA sequencing revealed that the GGI encodes a putative type IV secretion system. Type IV secretion systems include plasmid conjugation systems and virulence factor export systems and some type IV secretion systems carry out both processes. Mutations in the putative type IV secretion genes in the GGI result in loss of DNA secretion. These mutants also show delayed adherence to primary cervical cells and exhibit an unusual microcolony phenotype upon binding to cells. Mutations in atlA result in the same phenotypes as the other type IV secretion mutations as well as showing decreased PG-cytotoxin production. Thus AtlA is necessary for type IV secretion and is either directly or indirectly involved in PG-cytotoxin production. These results suggest that the GGI encodes an active type IV secretion system important in interaction of N. gonorrhoeae with the host. The goals of this proposal are contained in two specific aims. 1) We will make mutations designed to affect type IV secretion and test these mutants for secretion of proteins, DNA, and PG-cytotoxin. 2) We will test mutants with defects in secretion for phenotypes relevant to virulence, i.e., epithelial cell adherence and invasion, intracellular survival, and microcolony formation. Overall, these studies are designed to reveal the molecular mechanisms of this novel secretion system and identify previously unrecognized virulence factors important in gonococcal infection which may serve as new targets for chemotherapy or immunoprotection.